At least two types of mast cells are known. These include mast cells of the airway or lung and intestinal mucosa (“mucosal,” or MCT), and mast cells of the connective tissue including skin, lymph nodes and intestinal submucosa (MCTC). The majority of airway mast cells contain tryptase but lack chymase and carboxypeptidase A. Most connective tissue mast cells, by comparison, contain tryptase, chymase and carboxypeptidase A. Mast cells of different tissues can also be distinguished morphologically. See Bingham, C. O. et al., J. Allergy Clin. Immunol. 105:S527–S534 (February 2000), hereby incorporated by reference.
Mast cell precursors arise from the bone marrow, and enter the circulation as CD34+ mononuclear cells. After migrating to mucosal and submucosal sites in the airway and tissues, mast cell precursors undergo tissue-specific development into mature mast cells. Mature mast cells are characteristically heavily granulated. See Kempuraj, D. et al., Blood 93:3338–46 (May 15, 1999), hereby incorporated by reference.
The granules within mast cells contain preformed inflammatory mediators, including the serine proteases, tryptase and chymase, vasoactive substances such as histamine, and neuroactive agents such as serotonin and nerve growth factor. The rapid release of these preformed mediators is a process known as degranulation, a form of regulated exocytosis, and is generally initiated by an activation event, such as cross-linking of mast cell high-affinity IgE receptors (Fcε receptors), or stimulation by complement components, neuropeptides or other agents. Mast cell activation also leads to the synthesis and release of arachidonic acid metabolites and a variety of de novo synthesized cytokines, including tumor necrosis factor alpha (TNF-alpha), IL-5, and IL-13. See Church, M. and Levi-Schaffer, F., J. Allergy Clin. Immunol. 99:155–60 (February 1997); Bingham, C. O. et al., J. Allergy Clin. Immunol. 105:S527–S534 (February 2000), hereby incorporated by reference.
Mediator release from mast cells plays an important role in immediate and late-phase hypersensitivity, and in inflammation, allergy, parasite infection, and asthma. In the United States alone, over 50 million people suffer from asthma, rhinitis, or some other form of allergy. Therapy for allergy remains limited to blocking the mediators released by mast cells (anti-histamines), non-specific anti-inflammatory agents such as steroids and mast cell stabilizers which are only marginally effective at limiting the symtomatology of allergy. See Perou et al., J. Biol. Chem. 272(47):29790 (1997) and Barbosa et al., Nature 382:262 (1996), both of which are hereby incorporated by reference. Additionally, studies have implicated mast cells in the initiation and severity of neurologic disorders, including multiple sclerosis. See Secor, et al., J. Exp. Med. 191:813–21 (Mar. 6, 2000). Because of their ubiquitous distribution, mast cells are likely to participate in a variety of other conditions, including rheumatoid arthritis, inflammatory bowel disease, and interstitial cystitis. See Church, M. and Levi-Schaffer, F., J. Allergy Clin. Immunol. 99:155–60 (February 1997), hereby incorporated by reference.
Historically, high throughput screening for small molecule agents that effect mast cell activation has been limited by the inability of researchers to reproducibly generate or obtain large enough numbers of a single uniform population of mast cells necessary for conducting large scale screens. It has been very difficult to establish large numbers of these cells in culture due to their terminally differentiated character and the fact that they are frequently localized in specialized deep-tissue microenvironments. As such, purification of mature mast cells from a single donor is labor intensive and typically yields numbers of cells which are far too low for large scale small-molecule drug discovery or for traditional genetic screening.
Several growth factors and cytokines have been shown to be capable of stimulating various phases of mast cell development, maturation and/or activation. Stem cell factor (SCF), also called Steel factor, c-kit ligand, or mast cell growth factor, is known to support mast cell development, survival and function. At least one group has reported generating a population of 1015 mast cells by treating CD34+ cells with SCF over a period of over 50 weeks. See Kinoshita, et al., Blood 94:496–508 (Jul. 15, 1999), hereby incorporated by reference. The cultures thus established, however, were not homogenous in character, i.e., not exclusively mucosal mast cells. Furthermore, after two months the cells became increasingly tryptase/chymase positive, a phenotype characteristic of connective tissue-type mast cells, rather than mucosal mast cells. These methods also suffered from poor reproducibility by independent groups.
Several groups have reported that IL-6 enhances the SCF-induced development of human mast cells from CD34-positive cells. For example, Saito, et al., reported a slight enhancement of mast cell proliferation after 4–8 weeks of culture with both SCF and IL-6. See, Saito, H. et al., J. Immunol.157:343–50 (1996), hereby incorporated by reference. Kinoshita et al., supra, in contrast, reported that IL-6 inhibited the proliferation of SCF treated mast cells. Still others have reported that the addition of IL-6 to SCF treated mast cells merely promoted their survival. See Yanagida, M., et al. Blood 86:3705–14 (1995), hereby incorporated by reference. In general, reports from these groups indicated that prior methods have produced variable results and have yielded relatively small mast cell populations unsuitable for high throughput screening.
Basophils, like mast cells, arise from bone marrow and are heavily granulated. Basophils synthesize many of the same mediators as mast cells, and express the same high affinity Fcε receptors. Thus, basophils also mediate immediate hypersensitivity reactions to antigen. Additionally, basophils participate in cell-mediated hypersensitivity. Unlike mast cells, basophils mature in the bone marrow, from which they are recruited to tissue sites of inflammation. Basophils and mast cells can be further distinguished based on their surface marker expression patterns.
The tyrosine kinase receptor molecule flt-3 has a general tissue distribution. A ligand for flt-3 has been identified (flt-3 ligand). flt-3 ligand stimulates expansion of hematopoietic progenitor cells in bone marrow and spleen and stimulates mobilization of hematopoietic progenitor cells. See Robinson, et al., J. Hematother. & Stem Cell Res. 9:711–720 (2000).
Zhang et al. have demonstrated a synergistic effect of SCF and flt-3 ligand, when used with other factors, on in vitro expansion of umbilical cord blood cells. See Zhang, X. et al., Chin. J. Biotechnol. 15:189–94 (1999). However, the expanded cells of this report were not characterized phenotypically, and fully differentiated, functional mucosal mast cells were not described.
Accordingly, it is an object of the invention to provide methods of generating large uniform populations of mucosal, airway-type mast cells in vitro. Similarly, the invention also provides methods of generating large uniform populations of connective tissue-type mast cells and basophil cells.